The invention relates to methods and kits for detecting enzyme activity using bioluminescence. In particular, it relates to a novel assay system with increased light production for a sensitive and convenient detection of luciferase activity.
Bioluminescence is a naturally occurring phenomenon that has been utilized for a number of applications, particularly in molecular biology where the enzyme associated with it have been used as genetic reporters. Bioluminescence is nearly ideal for use as a genetic marker. Typically there is no endogenous luminescent activity in mammalian cells, while the experimentally introduced bioluminescence is nearly instantaneous, sensitive and quantitative. While numerous species exhibit bioluminescence, only a relative few have been characterized and cloned. Of these, only Firefly (Photinus pyralis) luciferase, Renilla luciferase and Aequorin have had much utility. Studies of the molecular components in the mechanism of firefly luciferases producing bioluminescence have shown that the substrate of the enzyme is firefly luciferin, a polyheterocyclic organic acid, D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ2-thiazoline-4-carboxylic acid (hereinafter referred to as “luciferin”).
Firefly luciferase is a monomeric 61 kD enzyme that catalyses the oxidation of luciferin in a two-step process, which yields light at 560 nm. The first step involves the activation of the carboxylate group of luciferin by acylation with the alpha-phosphate of ATP in the presence of magnesium to produce luciferyl adenylate with the elimination of inorganic pyrophosphate (PPi). In the second step, the luciferyl adenylate is oxidized with molecular oxygen to yield AMP, carbon dioxide and oxyluciferin. The oxyluciferin is generated in an electronically excited state. Upon transition to the ground state the oxyluciferin emits light.
The reaction scheme of the reaction hereinafter referred to a ‘luciferin-luciferase reaction’ is as follows:

Luciferase has many characteristics that make it ideal for a reporter enzyme. Its activity is not dependent on any post-translational modification, making it immediately available for quantitation. In addition, the luminescence is very bright, having very high quantum efficiency as compared to many other bio- and chemiluminescent reactions.
When light emission is initiated by the addition of luciferase into a reaction mixture containing ATP, Mg2+ (or an other divalent cation such as Mn2+), and luciferin, where all components are near or at saturating concentrations, one observes a rapid increase in light intensity followed by a rapid decrease in the first few seconds to a low level of sustained light intensity that may last hours. This rapid decrease in the rate of reaction has been thought to be due to product inhibition. These conventional “flash” type assays using firefly luciferase result in a flash of light, which decays rapidly with the addition of substrates to the enzyme. Especially in automated (e.g. robotic) assay procedures, this is a major problem as it dramatically reduces the time window in which a signal, if present, can be detected. Means for extending the duration of the light signal in the luciferase assay have been eagerly sought.
One approach, which achieved some popularity, to solving the problem of the kinetics of the luciferin-luciferase reaction and the associated difficulty of precisely measuring light emitted during the flash, was to use various inhibitors of the enzyme, which were reported to prevent the flash from occurring or to prolong light production. One such agent is arsenate. Arsenate lowers flash height and tends to prolong the light emission period. The decrease of the intensity of the light signal is considerable undesirable, in particular when microtiter plates or instruments capable of reading out strips are used. In addition, the use of arsenate is not desirable from an environmental point of view.
U.S. Pat. No. 4,246,340 likewise proposes a method for prolonging the light signal in a luciferin-luciferase assay based on the use of inhibitors, such as analogs of D-luciferin. This prolongs the light signal from seconds to a few minutes.
The cofactor coenzyme A (CoA) has been reported to affect the pattern of light emission in the luciferin-luciferase reaction. Airth et al., Biochimica et Biophysica Acta, vol. 27 (1958) pp. 519-532, report that, when CoA is added to a firefly luciferin-firefly luciferase reaction mixture, there is no effect on the initial peak of light intensity but luminescence will continue at a higher level for a time period that is proportional to the total CoA added. Airth et al. have shown that the total light emitted is greater in the presence of CoA than in its absence. U.S. Pat. No. 5,283,179 also discloses the addition of coenzyme A (CoA) in the assay reagent to yield greater enzyme turnover and thus greater luminescence intensity. This resulted in an increased light output that is maintained for at least 60 seconds.
It has been reported that other sulfhydryl compounds contribute to the stability of luciferases during preparation and storage of the enzymes. U.S. Pat. No. 4,833,075 discloses that dithiothreitol (DTT) will maintain luciferase activity at a level of 50% in an aged Photinus pyralis luciferase solution which, without the DTT, would have only 10% residual enzymatic activity compared to a freshly prepared luciferase solution. U.S. Pat. No. 4,614,712 describes that, when bacterial luciferase has been inactivated by disulfide formation, enzyme activity may be restored by addition of DTT, β-mercaptoethanol (β-ME), or other reducing agents.
U.S. Pat. No. 5,618,682 describes compositions and methods for increasing the duration of detectable photon emission of a luciferin-luciferase reaction. To that end, the reaction mixture containing luciferase, luciferin, ATP, and cofactors required for luciferase catalytic activity is mixed with a composition containing adenosine monophosphate, a radical scavenger (DITT) and a chelating agent (EDTA).
U.S. Pat. No. 5,650,289 reports that the combined use of CoA and DTT in the reaction mixture positively influences the kinetics of the luciferin-luciferase reaction. The half-life of the light signal, i.e. the time period after which 50% of the original light signal is observed, was assessed at 300 to 500 seconds.
It is an object of the present invention to provide an alternative luciferase detection assay system with improved light yield and extended-glow light emission for sensitive detection of firefly luciferase reporter enzyme.